Otitis media (OM) accounts for most bacterial respiratory infections in children in both developed and developing nations. Streptococcus pneumoniae (the pneumococcus) is the most common cause of childhood OM. Profound tissue inflammation and excessive secretion are the major hallmarks of pneumococcal OM. Mucosal inflammation and disease derived from pneumococcal infections are caused by high bacterial burden in the middle ear and other organs. Successful growth of S. pneumoniae in the infected organs/tissues directly depends on bacterial ability to withstand host immunity (immune evasion). However, the precise mechanisms underlying pneumococcal immune evasion are poorly understood. Our long-term objectives are: 1) to elucidate the molecular mechanisms of pneumococcal immune evasion and 2) to evaluate the potential of interfering with pneumococcal immune evasion as a strategy to prevent pneumococcal OM and other infections. The specific objective of this application is to determine how bacterial factors enhance S. pneumoniae resistance to host phagocytosis, a major host effector mechanism against Gram-positive pathogens. In the current grant period, we have successfully constructed and screened a mutant library of a low-passage multi-drug-resistant S. pneumoniae OM isolate in a chinchilla ear infection model. This study led to the discovery of 169 ear-infection-associated bacterial genes including the gene encoding the major surface protein CbpA of S. pneumoniae. Our subsequent studies show that CbpA inhibits complement-mediated phagocytosis of S. pneumoniae by recruiting the complement inhibitor factor H. Based on a mutant screening study in an in vitro phagocytosis model, we also found that the arcDT genes enhance pneumococcal antiphagocytosis by promoting capsule formation. These studies and other information have prompted us to hypothesize that S. pneumoniae possesses multiple mechanisms to evade host phagocytosis during the infection of the middle ear and other organs/tissues. We further postulate that interfering with these antiphagocytosis mechanisms can promote bacterial clearance and thereby prevent tissue inflammation and frank disease. The Specific Aims are: (i) to determine the structural basis of CbpA-mediated S. pneumoniae interactions with host factors; (ii) to determine how CbpA-mediated host-pathogen interactions promote S. pneumoniae infectivity in the middle ear; and (iii) to define how ArcDT promote capsule formation of S. pneumoniae. The data of the proposed studies will provide valuable information concerning the molecular mechanisms of pneumococcal immune evasion. Some of the immune-evasion factors may be attractive targets for development of inhibitory compounds and/or vaccines against pneumococcal OM and other infections. PUBLIC HEALTH RELEVANCE: The bacterium Streptococcus pneumoniae is a major causative agent of middle ear infection, bacterial pneumonia, blood infection, and meningitis. The current vaccine and treatment strategies have multiple limitations ranging from the limited vaccine coverage of virulent strains to rising antibiotic resistance of the pathogen. Profound tissue inflammation and excessive secretion are the major hallmarks of S. pneumoniae disease. Mucosal inflammation and disease derived from S. pneumoniae infections are directly caused by high bacterial burden in the infected tissues. Successful growth of S. pneumoniae in the host environments depends on bacterial ability to withstand host defense mechanisms (immune evasion). However, the precise mechanisms underlying S. pneumoniae immune evasion are poorly understood. Our long-term objectives are: 1) to elucidate the molecular mechanisms of S. pneumoniae immune evasion, and 2) to evaluate the potential of interfering with S. pneumoniae immune evasion as a strategy to prevent S. pneumoniae disease. This project will characterize S. pneumoniae genes that are involved in enhance bacterial resistance to host phagocytosis, a process for uptake and killing of pathogens by host immune cells. The information will significantly enhance our knowledge in disease pathogenesis of S. pneumoniae. The bacterial genes identified in this project can be targeted to treat and prevent S. pneumoniae disease by disarming the immune evasion arsenals of S. pneumoniae through novel anti-bacterial drugs and vaccines.